Immunoblotting Technique for Protein Analysis

Immunoblotting was performed as described in the previous study^{29 (link)}. Briefly, cells were washed once with PBS and lysed with modified RIPA buffer (10 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1 mM EGTA, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS) containing phosphatase (1 mM NaF and 1 mM Na₃VO₄) and protease inhibitors. Proteins were extracted on ice with vortexing three times for 30 min and lysates were centrifugated at 10,000×g for 10 min at 4 °C. The supernatants were used for immunoblotting after boiling in 1 × SDS-sample loading buffer with reducing agent at 97 °C for 7 min. Protein concentration was analyzed by the BCA method (Sigma). Each protein sample (20–40 μg) was electrophoresed on 4–12% NuPAGE Bis–Tris Protein gels (Invitrogen) at a constant current of 20 mA, followed by transfer to NC (nitrocellulose) membranes (GE Healthcare). A membrane of protein blots from various human normal tissues (TB37-Set-1) was purchased from G-Biosciences. Each membrane was blocked with 5% skim milk, followed by immunoblotting with the indicated antibodies. The blots were developed using chemiluminescence (Thermo Scientific), and the images were analyzed using the ImageJ software.